Development of an asporogenic Bacillus licheniformis for the production of keratinase

AIMS: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. METHODS AND RESULTS: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. CONCLUSIONS: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.     

Disruption of the gene encoding the secreted acid protease (ACP) in the yeast Candida tropicalis

The gene for the secreted acid protease (ACP), a potential virulence factor of Candida species, was inactivated in Candida tropicalis by gene disruption. The disruption was performed by cotransformation of an ade2 C. tropicalis mutant with a linear DNA fragment carrying a deletion in ACP, and the replicative vector pMK16 which carries a selectable ADE2 gene marker. Few of the transformants exhibited lower protease secretion levels and were shown to have one deleted and one unaffected ACP copy, since C. tropicalis is a diploid yeast. These transformants were rendered homozygotic for this deletion by mild UV-treatment. One of the homozygotic acp deletion mutants obtained was completely devoid of extracellular protease activity and grew poorly on bovine serum albumin-containing medium. This mutant could be complemented by an ACP fragment inserted in pMK16, but also by an acid protease gene isolated from C. parapsilosis.     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

Secretion by Pseudomonas aeruginosa: fate of a cloned gram-positive lipoprotein deletion mutant.

In gram-positive organisms, glyceride-cysteine thioether lipoproteins are frequently associated with secretion. They constitute membrane-bound forms retained by the cell but releasable late in growth phase. Most gram-negative organisms secrete very few proteins to the culture fluid; thioether lipoproteins in such organisms, typified by the enteric bacterium Escherichia coli, are integral outer membrane components for the most part. Unusual among gram-negative organisms, however, are Pseudomonas strains, known for extracellular export of a number of proteins. To examine whether a fundamental difference exists between the processing of lipoproteins in Pseudomonas strains and in nonsecretory gram-negative organisms, we examined the fate in Pseudomonas aeruginosa and E. coli of a cloned gram-positive secretory lipoprotein, Bacillus licheniformis penicillinase. A nonlipoprotein deletion mutant of the same gene was also examined in P. aeruginosa, and its processing was compared with that in E. coli. No important differences were found between P. aeruginosa and E. coli for either the lipoprotein or its deletion mutant. Thus, the contrast in secretory abilities of the two organisms does not appear to result from a difference in their general secretory systems.     

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.     

The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases.

Two Escherichia coli mutants that lack both cytochrome o and d terminal oxidases are able to grow with glucose as the carbon source but not with the aerobic substrates succinate or lactate. One of these, GV101, is a deletion mutant of cytochrome o and a point mutation of cytochrome d. The other, GK100, is a total deletion mutant of all the genes for both cytochromes. When these mutants were transformed with a plasmid containing the gene for the bacterial hemoglobin from Vitreoscilla, they were capable of growth in the presence of succinate or lactate and showed aerobic respiration in the presence of these substrates, unlike the parent strains. Cells transformed with a plasmid containing the gene for the hemoglobin but lacking the native promoter did not express the hemoglobin and did not respire. Membrane vesicles prepared from the cells consumed oxygen in the presence of succinate. This succinate-supported respiration decreased with successive washings of the vesicles but was restored by adding E. coli cytosol containing the hemoglobin or by adding the hemoglobin purified from Vitreoscilla. This respiration was inhibited by cyanide.     

Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain.

Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids.     

Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence

Vibrio vulnificus, a halophilic estuarine bacterium, causes a fatal septicemia and necrotizing wound infection. To investigate the role of cAMP in V. vulnificus virulence regulation, an in-frame deletion mutant of the cya gene encoding adenylate cyclase was constructed. The cya null mutation resulted in a pleiotropic change of virulence phenotypes. The production of hemolysin and protease, the motility, and the cytotoxicity were decreased by the cya mutation. The defects in the cya mutant were functionally complemented in trans by a plasmid carrying the wild type cya allele. The V. vulnificus cya mutant exhibited a 100-fold increase in LD50 to mice. The result indicates that cAMP plays an essential role in the global regulation of V. vulnificus virulence.     

强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株。为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善。利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称的位置引入耶氏解脂酵母来源的脂肪酶基因lipY2。整合菌株在摇瓶发酵44h时,木聚糖酶、脂肪酶酶活力分别达(58±2.07)U/mL和(207±10.62)U/mL,其分泌表达促进了地衣芽孢杆菌对发酵培养基的分解与利用,提高了培养基中还原糖、上清总氮的含量和沉淀中含氮化合物的分解;细菌生物量较地衣芽孢杆菌原始菌株提高了11.76%,同时碱性蛋白酶的发酵周期较原始菌提前了4h,碱性蛋白酶产量提高了14.41%。地衣芽孢杆菌2709分泌酶系的丰富和发酵性能的改善为在饲料行业中作为微生物制剂的地衣芽孢杆菌提供了改造的方法。     

Eglin C与2709碱性蛋白酶相互作用分析及亲和纯化方法

Eglin C是来自水蛭中的一种小型热稳定蛋白质,属于马铃薯胰凝乳蛋白酶抑制剂家族,可以抑制弹性蛋白酶、枯草杆菌蛋白酶、组织蛋白酶、α-lytic蛋白酶以及胰凝乳蛋白酶等。然而,利用eglin C纯化蛋白酶,尚未见研究报道。本文将化学合成的编码 eglin C及其突变体的基因序列,克隆到原核表达载体pQE30,在大肠杆菌BL21获得His6-Tag-eglin C及其突变体的重组蛋白质。SDS-PAGE显示,eglin C蛋白的分子量大约8 kD。His6-Tag-eglin C对胰凝乳蛋白酶、地衣芽孢杆菌2709碱性蛋白酶、枯草芽孢杆菌PB92碱性蛋白酶、枯草杆菌中性蛋白酶的半抑制剂浓度（IC50）分别为0.20±0.15、0.24±0.19、3.33±0.47和52.46±0.38 μmol/L。利用分子对接、基因突变以及荧光光谱等,分析eglin C及其突变体与2709蛋白酶的相互作用。结果显示,2709碱性蛋白酶对eglin C荧光淬灭属于静态淬灭,解离常数为2.60×10-7 mol/L,eglin C中的Asn50 残基对eglin C和2709碱性蛋白酶的结合发挥重要作用。利用eglin C与蛋白酶的特异结合的特性,构建亲和纯化载体,用于纯化来源于地衣芽孢杆菌的2709碱性蛋白酶,相比常规的蛋白酶纯化,显著简化了操作步骤。     

地衣芽胞杆菌碱性蛋白酶基因克隆与表达特性

目的建立高产量和高活力的地衣芽胞杆菌碱性蛋白酶基因表达体系。方法采用PCR技术克隆获得目的基因,将其连入表达质粒pET-32 a构建原核表达重组质粒,经测序鉴定后,转化BL21大肠埃希菌,不同温度下IPTG诱导表达融合蛋白,测定酶活;进一步对该基因和编码蛋白进行同源性比较和酶学性质分析。结果碱性蛋白酶基因序列全长1 149 bp,编码382个氨基酸,同源性为99%,融合蛋白分子质量为62 kD,蛋白酶酶活为29 000 U/mL,并且在25℃时是以可溶蛋白形式表达,37℃时部分蛋白以包涵体形式存在。结论此种表达体系可以成功表达具有生物活性的碱性蛋白酶,诱导温度对蛋白酶存在形式具有较大影响。     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation

Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.     

Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli

The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.     

Role of autolysins in the killing of bacteria by some bactericidal antibiotics

The rapid lysis of Bacillus licheniformis NCTC 6346 and B. subtilis 168 trp caused by vancomycin and d-cycloserine can be inhibited by stopping protein synthesis. Protein synthesis must be stopped for more than one doubling time of the cells before addition of wall inhibitors. Poorly lytic mutants (lyt(-)) of B. licheniformis required 10 to 20 times the concentration of vancomycin or cycloserine to be added to growing cultures to cause even slow lysis. At lower concentrations growth of the mutants is stopped, but the bacteria remain fully viable. Sensitivity of mucopeptide synthesis to vancomycin is the same in both mutants and parent. Sensitivity to the action of d-cycloserine is slightly less in the mutant than in the parent.     

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.     

The thiM locus and its relation to phosphorylation of hydroxyethylthiazole in Escherichia coli.

A mutant of Escherichia coli lacking hydroxyethylthiazole kinase (EC 2.7.1.50) was produced by a further mutation of a temperature-sensitive, auxotrophic mutant for hydroxyethylthiazole. The parent cells possessed two distinct enzymes capable of phosphorylating hydroxyethylthiazole: one was hydroxyethylthiazole kinase, and the other was a phosphotransferase species that required p-nitrophenylphosphate as a phosphoryl donor. Osmotic shock fluid prepared from the mutant cells phosphorylated hydroxyethylthiazole to an extent comparable to that observed with shock fluid from the parent cells, whereas extracts from shocked cells were unable to catalyze the kinase reaction. Shock fluid from a mutant of the other type obtained as a reduced phosphatase activity against p-nitrophenylphosphate did not show any appreciable activity for the phosphotransferase reaction, while extracts from shocked cells showed full kinase activity. The former mutant had lost its ability to grow on hydroxyethylthiazole at high temperature, but the latter mutant still responded to it. It thus appears that the kinase is an enzyme which might play a role in the biosynthesis of thiamine PPi in situ. By conjugation and P1 transduction, a gene governing hydroxyethylthiazole kinase activity, for which we propose the designation thiM, was mapped on the chromosome close to thiD, a gene specifying phosphomethylpyrimidine kinase activity.     

烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌YP1产胞外蛋白酶的影响

考察了添加5%(V/V)浓度的正庚烷、正辛烷、正癸烷、十二烷、十四烷、十六烷等烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌(Bacillus licheniformis)YP1的生长及产胞外蛋白酶的影响.结果表明5%(V/V)浓度的各种烷烃溶剂对YP1蛋白酶的稳定性及菌体生物量均无显著影响,正庚烷、正辛烷、正癸烷等溶剂显著抑制YP1产蛋白酶,而十二烷、十四烷,十六烷能提高YP1产蛋白酶1倍以上.发酵液中十四烷的浓度(1%-8%,VIV)与蛋白酶的活力呈正相关性,添加十四烷后发酵过程中蛋白酶活力的显著增加出现在菌体生长的对数后期.培养过程中添加十四烷能导致YP1菌体形态显著变小.首次报道了烷烃溶剂对极端微生物产蛋白酶的影响.     

地衣芽胞杆菌ATCC9945A中γ-聚谷氨酸降解酶基因的克隆、表达及降解性能鉴定

目的:验证在地衣芽胞杆菌ATCC9945A中存在着γ-聚谷氨酸降解酶基因(ywtD),为下一步解决在γ-聚谷氨酸微生物发酵合成过程中产物γ-PGA降解的技术性难题提供依据。方法:通过在pET-28b(+)大肠杆菌表达系统克隆表达地衣芽孢杆菌ATCC9945A中的ywtD基因,对诱导表达条件进行优化,采用SDS-PAGE和Western Blot方法检测目的蛋白的表达,并体外酶解实验验证其活性。结果:PCR扩增得到了一个1 245bp的基因片段,预期编码414个氨基酸,诱导表达后得到一个分子量大小约为45.6 kDa的表达产物。Western Blot分析结果表明ywtD基因得到了有效表达。体外酶解实验表明该表达产物具有降解γ-PGA的活性。结论:证明在地衣芽胞杆菌ATCC9945A中存在着γ-聚谷氨酸降解酶基因。